Vegetable starches, e.g., cornstarch, are widely used in the industrial manufacture of products such as syrups and biofuels. For example, high fructose corn syrup (HFCS) is a processed form of corn syrup having high fructose content and a sweetness comparable to sugar, making HFCS useful as a sugar substitute in soft drinks and other processed foods. HFCS production currently represents a billion dollar industry. Similarly, the production of ethanol from vegetable starches is a rapidly expanding industry. Ethanol has widespread applications as an industrial chemical, a gasoline additive, or a liquid fuel by itself. The use of ethanol as a fuel or fuel additive significantly reduces air emissions while maintaining or even improving engine performance. On the other hand, ethanol is a renewable fuel, so that its use may reduce dependence on finite fossil fuel sources. Furthermore, use of ethanol may decrease the net accumulation of carbon dioxide in the atmosphere.
Syrups and biofuels can be produced from starch by an enzymatic process that catalyzes the breakdown of starch into glucose. This enzymatic process typically involves a sequence of enzyme-catalyzed reactions:
(1) Liquefaction: Alpha-amylases (EC 3.2.1.1) first catalyze the degradation of a starch suspension, which may contain 30-40% w/w dry solids (ds), to maltodextrans. Alpha-amylases are endohydrolases that catalyze the random cleavage of internal α-1,4-D-glucosidic bonds. Because liquefaction typically is conducted at high temperatures, e.g., 90-100° C., thermostable alpha-amylases, such as an alpha-amylase from Bacillus sp., are preferred for this step. Alpha-amylases currently used for this step, e.g., alpha-amylases from B. licheniformis (AmyL), B. amyloliquefaciens, and Geobacillus stearothermophilus (AmyS), do not produce significant amounts of glucose. Instead, the resulting liquefact has a low dextrose equivalent (DE) and contains maltose and sugars with high degrees of polymerization (DPn).
(2) Saccharification: Glucoamylases and/or maltogenic alpha-amylases catalyze the hydrolysis of non-reducing ends of the maltodextrans formed after liquefaction, releasing D-glucose, maltose and isomaltose. Saccharification produces either glucose-rich or high-maltose syrups. In the former case, glucoamylases typically catalyze saccharification under acidic conditions at elevated temperatures, e.g., 60° C., pH 4.3. Glucoamylases used in this process typically are obtained from fungi, e.g., Aspergillus niger glucoamylase used in Optidex® L400 or Humicola grisea glucoamylase. De-branching enzymes, such as pullulanases, can aid saccharification.
Maltogenic alpha-amylases alternatively may catalyze saccharification to form high-maltose syrups. Maltogenic alpha-amylases typically have a higher optimal pH and a lower optimal temperature than glucoamylase, and maltogenic amylases typically require Ca2+. Maltogenic alpha-amylases currently used for this application include B. subtilis alpha-amylases, plant amylases, and the alpha-amylase from Aspergillus oryzae, the active ingredient of Clarase® L. Exemplary saccharification reactions used to produce various products are depicted below:

(3) Further processing: A branch point in the process occurs after the production of a glucose-rich syrup, shown on the left side of the reaction pathways above. If the final desired product is a biofuel, yeast can ferment the glucose-rich syrup to ethanol. On the other hand, if the final desired product is a fructose-rich syrup, glucose isomerase can catalyze the conversion of the glucose-rich syrup to fructose.
Saccharification is the rate-limiting step in the production of a glucose-rich syrup. Saccharification typically occurs over 48-72 hours, by which time many fungal glucoamylases lose significant activity. Moreover, it takes a significant portion, e.g., more than 70%, of saccharification time for the glucose yield to increase from 85% to 96%. This is mainly due to the inefficient hydrolysis of low molecular weight oligosaccharides by the glucoamylase. Accordingly, maximization of glucose production would require a relatively high dose of glucoamylase and/or a longer saccharification period. Furthermore, although maltogenic alpha-amylases and glucoamylases both can catalyze saccharification, the enzymes typically operate at different optimal pH and temperatures, as shown above. If both enzymes are used sequentially, the difference in reaction conditions between the two enzymes necessitates adjusting the pH and temperature, which slows down the overall the process and may give rise to the formation of insoluble amylose aggregates.
Accordingly, there is a need in the art for an improved starch processing method to make industrial products. In particular, there is a need for improved efficiencies in a saccharification step.